Two giant muscle proteins Mr about 10 6) have been discovered recently, titin and nebulin, that constitute about 10-15% of the myofibrillar protein mass. The broad long-term goal of this proposal is to study the function of these proteins and understand their role in muscle disease. Recent studies indicate that titin in vertebrate muscle and minititin, the analogous protein found in insect flight muscle (IFM), may develop passive force. Passive force is an important, but not well investigated factor in muscle. This importance is especially striking in cardiac muscle where passive force is part of the diastolic wall tension that determines the extent of filling of th heart and its subsequent stroke volume. Striking also is the recent finding that passive force in insect IFM varies linearly with active force, indicating that passive force regulates cross-bridge interaction (Granzier and Wang, 1993). Thus, the study of titin and minititin provides a basis for understanding the molecular mechanism that underlies passive force, and how this force interacts with the actomyosin system. Main experiments: Aim 1) Investigate the mechanism that underlies the interplay between passive tension and actomyosin interaction in insect IFM. The effect of passive tension will be investigated on the rate constant for the transition of cross-bridges from force-generating-states to non- force=generating states (gapp) and on the rate constant for the return to force-generating-status (fapp); Aim 2) Does interplay between passive tension and actomyosin interaction occur in cardiac and skeletal muscle? This will be investigated by studying the effect of passive tension on weak and strong cross-bridges in cardiac myocytes and skeletal muscle fibers (cf. Granzier & Wang, 1993); Aim 3) Study the interaction between titin and the other filaments in the sarcomere. (a) What is the nature of interaction between titin and the thick filaments? (b) Does the titin interact with thin filaments? It is expected that the proposed studies into the function of titin and nebulin will lead to a better fundamental understanding of the physiology of cardiac and skeletal muscles. and that this will improve the foundation for understanding various myopathies of skeletal and cardiac muscle.